The present invention is directed to enzymatic methods for preparing glycerides and to designed glycerides of specific composition.
The present invention is, in part, directed to margarine oils, and more particularly is directed to margarine oils having both low trans- acid content and low content of intermediate chain saturated fatty acids, together with margarine-type thermal melting characteristics and/or smooth organoleptic consistency. The invention is also directed to oils having a very low level of saturated fatty acid components, and to enzymatic transesterification methods for producing low-saturate, edible oil products. This invention may also be used to introduce specific health-promoting fatty acids, e.g., omega-3 fatty acids such as eicosapentanoic acid, into triglyceride oils and fats. This invention may also be used to introduce medium chain fatty acids, e.g., having six to twelve carbon atoms per molecule, into triglycerides. Said fatty acids can be derived from natural or synthetic sources and include both odd and even carbon numbers. This invention can also be used to incorporate difunctional fatty acids, such as succinic, adipic, octanedioic acid, etc. so as to create glyceride oligomers which may have desirable properties as cooking oils, etc. This invention may also be used to introduce incompletely metabolized, low calorie fatty acids, e.g., furan fatty acids, derived from natural or synthetic sources into triglyceride oils and fats. In addition, the invention is directed to counter-current methods for enzymatic esterification, interesterification, transesterification and refining.
Margarine oils are predominantly mixtures of triglycerides which have a plastic consistency at refrigeration and/or ambient temperature, but which have the essential characteristic of melting readily and with substantial completeness in the mouth of the consumer. Such a melting characteristic requires a solid fat index which has an extended gradient over a broad temperature range. In addition, margarine oils must have a crystal size and shape which provides a smooth organoleptic consistency without graininess or similar mouthfeel defects in homogeneity. Margarine oils are distinguished from plastic shortenings, which typically have a high stearic acid content and a melting point higher than body temperature, which is characterized by the presence of substantial solid fat content at body temperature. The high solid fat content at body temperature is desirable in plastic shortenings for use in various hot dishes, or in dispersed form in products such as pastries and baked goods where the high solid fat content is not deleterious. However, a residue of solid fat such as that present in conventional plastic shortenings which fails to melt at body temperature, imparts to a margarine oil an unacceptable waxy sensation in the mouth. As used herein, the term "margarine oil" and "margarine fat" are used interchangeably.
Coconut oil and the other oils of the lauric acid type have a relatively low melting profile as a result of their relatively high concentration of intermediate chain saturated fatty acids and may conventionally be utilized as a component of margarine oils. By "intermediate chain saturated fatty acid" is meant an edible saturated fatty acid having from 8 to 16 carbon atoms, particularly including palmitic, myristic and lauric acids or mixtures thereof. Because the melting points of saturated fatty acids exhibit a progressive increase as the carbon chain is lengthened, fats of the coconut oil type which contain relatively large proportions of C.sub.8 to C.sub.16 saturated fatty acid moieties, have lower melting points than fats with an equivalent degree of unsaturation that comprise a high proportion of C.sub.18 fatty acid glycerides. However, diets high in intermediate chain saturated fatty acids, notably lauric, myristic and palmitic acids common to lauric acid oils, have been reported in the medical literature as being a factor in the production of plasma cholesterol in populations at risk for coronary heart disease. However, stearic acid although it is a saturated fatty acid, has been reported to have minimal or even reducing effect on cholesterol level ["Effect of Dietary Stearic Acid on Plasma Cholesterol and Lipoprotein Levels", Bonanome, et al., New England Journal of Medicine, Vol. 318, 1244-1271 (1988)]. Accordingly, although lauric acid oils have desirable margarine oil properties, margarine oils which have low intermediate chain fatty acid content would be desirable.
Vegetable oils, such as cottonseed, peanut, sesame, corn and sunflower oils, and other liquid oleic-linoleic acid oils, as well as soybean oil, may be partially hydrogenated for the production of margarine oils of the requisite melt and consistency characteristics of broad thermal melting range, substantially complete melting at body temperature, and smooth organoleptic characteristics. The desired consistency is typically obtained by blending two or more partially hydrogenated vegetable oils, or blending liquid (unhydrogenated) vegetable oil with a partially hydrogenated vegetable oil. However, conventional partial hydrogenation of vegetable oils containing unsaturated acids, depending on catalyst selectivity, degree of hydrogenation and other processing variables, may produce substantial amounts of unsaturated fatty acids of trans-, rather than cis- configuration. margarine oils which contain minimal amounts of such trans- acid moieties, together with the requisite solid fat index thermal profile, smooth organoleptic consistency and low intermediate chain fatty acid content, would be desirable.
The main components of margarine oils are triacylglycerols (triglycerides) which are triesters of glycerol and various saturated and unsaturated fatty acids. The physical properties of fats and oils are, to a large extent, determined by the characteristics of the individual fatty acid moieties and by their distribution within the triglyceride molecule. Interesterification is a technique which may be used to alter the fatty acid composition and distribution and therefore the physical properties of triglyceride mixtures. In such processes, chemical catalysis by sodium metal or a sodium alkoxide is used to promote the migration of fatty acyl groups between and within glycerol molecules, so that the product consists of acylglycerol mixtures in which the fatty acyl groups are randomly distributed among the glyceride molecules. The use of enzymes such as site specific lipases permits formation of novel, functional fats which cannot be obtained by conventional chemical processes.
Edible oils and fats typically primarily comprise various fatty acid triesters of glycerol with the structure of the fatty acid moieties and their distribution on the glycerol backbone determining the physical characteristics of the oil or fat. The specific types of fatty acids also play an important role in diet and health. Fats and oils in general are a rich source of energy in the diet and are important in the synthesis of membranes and other essential cell components. Moreover, dietary fatty acid content may potentially be controlled to affect physiological characteristics such as serum cholesterol levels. For example, studies of normcholesterolemic men has shown that a dietary decrease in saturated fatty acids may have more of an effect in lowering serum cholesterol [Keys, "Prediction of Serum Cholesterol Response to Change in Fats in the Diet" , Lancet, 2:959-962] than an increase in polyunsaturated fatty acids.
Natural vegetable oil triglycerides typically contain substantial amounts of esterified saturated fatty acids. For example, soybean oil may typically contain about 14-16 weight percent of esterified saturated fatty acids, and natural canola oil may contain about 5-8 weight percent of esterified saturated fatty acids. Intermediate carbon chain length (i.e., C.sub.12 -C.sub.16) dietary saturated fatty acids, notably lauric, myristic and palmitic acids, have been reported in the medical literature as being a more significant factor in the increase of plasma cholesterol than stearic acid, which has been reported to have minimal or even reducing effects on cholesterol levels ["Effect of Dietary Stearic Acid on Plasma Cholesterol and Lipoprotein Levels", Bonanome, et al., New England Journal of Medicine, Vol. 318, 1244-1271 (1988)]. Soybean oil and canola oil typically contain, respectively, over 10 percent and over 3 percent by weight of esterified intermediate chain length saturated fatty acids, primarily palmitic acid. Accordingly, it would be desirable to economically produce or manufacture triglyceride oils having very low levels of saturated fatty acids, and particularly low levels of lauric, myristic and palmitic acids. Most food products prepared from vegetable oils having less than about 3.5 weight percent of esterified saturated fatty acids may be regarded as substantially free of such fatty acids for regulatory purposes.
Potentially, the modification of vegetable oils to produce low-saturate oil products could be carried out by dehydrogenation, chemical transesterification, enzymatic transesterification or genetic selection and modification. However, dehydrogenation processes are not available for selectively dehydrogenating esterified saturated fatty acids of vegetable oils. The use of somaclonal variation as a means of selection for genetic variation in plant parts which may produce triglycerides with specific desirable fatty acids may be used, but problems may arise in the stabilization of the desired trait in future generations. Recombinant DNA techniques might be used to increase the production of an oil of predetermined composition, but this is a very complex task in difficult or presently unknown areas of plant lipid biosynthesis. [Stumpf, "Biosynthesis and Function of Plant Lipids", Am. Soc. Plant Physiol., pp. 1-15, 1983]. For example, fatty acid synthesis has many potential rate limiting enzymes as well as proteins, such as acetyl-CoA carboxylase, ACP-acetyl transferase, 3-oxoacyl ACP synthase, ACP malonyl transferase and acyl carrier protein. Modifications of specific triglyceride synthesis entail changes in fatty acid composition dependent on acyl ACP thioesterase and various desaturase complexes, as well as acyltransferase enzymes which attach the fatty acid moieties to glycerol. Thus, to isolate the rate limiting enzymes/proteins and their corresponding genes to create transgenic plants which specifically express them in tissues for storage purposes poses substantial technical problems.
Chemical and enzymatic transesterification are well known for modifying the fatty acid composition or distribution of triglyceride oils. Chemical transesterification is based on the use of a chemical catalyst such as sodium methoxide or a sodium metal to promote the migration of the fatty acid moieties between glyceride molecules, to produce a random distribution of the fatty acid moieties.
Enzymatic transesterification of triglycerides may also be used to modify the characteristics and/or composition of triglycerides. Such processes may be used for selective interchange under relatively mild reaction conditions. For example, vegetable oils may be transesterified with a fatty acid or lower alkyl monoester to produce a variety of end products as described in U.S. Pat. Nos. 4,268,527; 4,426,991; 4,275,011; 4,472,503 and U.K. Application 2,199,397.
Extracellular microbial lipases are generally of three types, depending upon their specificity. One group of lipases is generally nonspecific, both as regards the position on the glycerol molecule which is hydrolyzed or esterified, and the nature of the fatty acid released or esterified. Depending on the reaction conditions, such lipases catalyze the nonselective hydrolysis, alcoholysis and/or esterification (including transesterification) of fatty acid triglycerides. The lipases produced by Candida cylindracae, also known as C. rugosa (Benzonana, G. and S. Esposito, Biochim. Biophy. Acta. 231:15 (1971)), Corynebacterium acnes, (Massing, G. S., Ibid. 242:381 (1971)), and Staphylococcus aureus, (Vadehra, D. V., Lipids 9:158 (1974)), Candida lipolytica and Pseudomonas fluorescens are examples of such nonspecific lipases.
A second group of lipases preferentially acts on the primary, 1- and 3-positions of the glycerol or triglyceride molecule. When a 1-, 3-positionally specific lipase is used to catalyze the transesterification of a mixture of triglycerides or a mixture of triglyceride plus free fatty acid or monoester, the action of the enzyme is substantially confined to the 1- and 3-positions of the glycerol. The lipases of Rhizopus delemar and Mucor miehei such as described in U.S. Pat. No. 4,798,793, are examples of 1-,3- specific lipases, as are the lipases of Aspergillus niger, Rhizopus arrhizus, Rhizopus niveus, Muror javanicus, Rhizopus javenicus, Rhizopus oxyzae.
A third group of lipases has substantial selectivity for certain long chain unsaturated fatty acids having a cis- double bond at the 9-position from the carboxylate group of the fatty acid. Long chain saturated fatty acids, and unsaturated fatty acid esters without a double bond in the 9-position, are only slowly hydrolyzed in the presence of such lipases. Thus the esters of oleic, palmitoleic, linoleic and linolenic acids, all of which have a cis double bond in the 9-position, are preferentially hydrolyzed, esterified or transesterified. The presence of an additional double bond between the carboxyl group and the double bond in the 9-position makes fatty acid esters resistant to the action of this lipase. Triglycerides containing medium chain saturated C.sub.10 and C.sub.8 fatty acids may exhibit some, albeit reduced, reactivity with such enzymes. Examples of such delta-9 specific lipases which preferentially act on long-chain fatty acids containing a cis- double bond in the 9-position are the lipase produced by the mold Geotrichum candidum [Macrae, A. R., in Microbial Enzymes and Biotechnology, edited by W. M. Fogarty, Applied Science Publishers, London, 1983, p. 225, Jensen, R. G., Lipids 9:149 (1974), Jensen, R. G., and R. E. Pitas, in Lipids, edited by R. Paolette, G. Porcellati and G. Jacini, Raven Press, New York, 1976, Vol. 1, p. 141], and the lipase produced by Penicilliun cyclopium [Glyceride Synthesis by Four Kinds of Microbial Lipase, Tsujisaka, et al.; Biochim. Biophy. Acta. 489; 415-422 (1977)]. Such lipases will activate transesterification of unsaturated delta-9 fatty acid groups of glyceride oils, but do not affect the saturated acid components of the oils.
Enzymatic methods which may be used to reduce the saturated fatty acid content of vegetable oils to levels below about 3.5 weight percent, and total levels of intermediate chain length fatty acids below about 2 weight percent, would be desirable, and it is an object of the present invention to provide such low-saturate vegetable oils. It is a further object to provide such methods which may be used to provide low-saturate oils having specific unsaturated fatty acid distribution, as well as low saturate edible oils having specific, nutritionally desirable properties of oleic, linoleic and linolenic acids. These and other objects will become apparent from the following detailed description and the accompanying drawings.
Egg yolks provide excellent functional emulsification properties for food products such as mayonnaise, and are a necessary or desirable component for many food products such as spoonable and pourable food dressings. The functional emulsification properties of egg yolks are believed to be largely attributable to phosphatide, protein and phosphatide/protein complex components of the egg yolk. However, in addition to these components which provide functional emulsifying properties, egg yolks also contain a substantial amount of cholesterol.
Methods are desirable for removing cholesterol from egg yolks without solvent denaturation or heat-denaturation of the egg yolk protein, and without causing substantial loss of phosphatides or phosphatide/protein complexes which provide the desirable functional characteristics of egg yolks. Methods which also could optionally be carried out to minimize the loss of natural egg yolk triglycerides would also be desirable. It would also be desirable if such methods could be provided which could be utilized to separate cholesterol from dry or liquid whole egg products without denaturation of egg protein.
A wide variety of methods have been used for removal of cholesterol from egg yolks, including hexane and aqueous alcohol-hexane extraction, liquified methyl ether extraction, and supercritical carbon dioxide extraction, but each of these methods has characteristic limitations or disadvantages. Extraction of egg yolk cholesterol into vegetable oil is effective for providing substantially cholesterol-free or cholesterol reduced egg yolks, but this produces a stream of cholesterol-containing vegetable oil. Such vegetable oil could be reused or used as a more desirable transesterification source material if the cholesterol could be readily removed.
Accordingly, it is an object to provide novel methods for separation of cholesterol from egg yolks and whole eggs. It is a further object to provide methods for producing low-cholesterol or cholesterol free food products such as mayonnaise, spoonable dressings and pourable dressing. These and other objects will be apparent from the following detailed description and the accompanying drawings.